1. Field of the Invention
Embodiments of the invention relate generally to the field of biological and/or chemical sensing. More particularly, embodiments of the invention relate to electrically active combinatorial-chemical (EACC) chips for biochemical analyte detection.
2. Background Information
Currently, biological and chemical analyte detections are based primarily on specific interaction between analytes and their binding partners. To perform high throughput assays, a large number of molecular probes need to be immobilized on a surface to form a microarray. Such microarrays are sometimes referred to as bio-chips (e.g., protein chips or gene chips). Preparing a large number of specific polymeric probes (e.g., antibodies or nucleic acids) is, however, both time-consuming and costly. Moreover, immobilizing the polymeric probes in discrete small surface areas is technically difficult and expensive. It is desired to have a more efficient approach to preparing and immobilizing probes.
Traditional approaches to making biochips involve chemically preparing polymeric probes and then subsequently spotting the chemically prepared polymeric probes on the chips. However, the minimum feature size attainable with these probes is typically >100 um for a protein chip (array), or >1 um for a gene chip (array). It is desired to have smaller feature sizes available in the future. While higher density bio-chips are clearly desirable from the perspective of both cost to manufacture and clinical efficiency, fabricating higher density bio-chips based on smaller polymeric probe feature sizes is both technically challenging and time-consuming. It is desired to have an approach that will permit the fabrication of chips based on smaller probe feature sizes.
Referring to FIGS. 1A and 1B, current biochips for direct analyte detection (antibody chips, DNA chips, aptamer chips) are based on interactions of analytes with their polymeric binding partners (probes), each of the latter of which presents unique intra molecular binding sites. Referring to FIG. 1A, a binding partner (probe) 110 is immobilized on a substrate 120. The binding partner 110 then binds with an analyte 130, thereby enabling the detection of the analyte 130. This binding approach is based on the principle of using a single, unique and large molecule for specific binding of analytes. This approach is highly specific and accurate, and generally involves small dimension(s). On the other hand, this approach is very costly and time-consuming because of the need to obtain analyte-specific probes or binding partners, and is generally inflexible. Also, as only known probes are used to detect known analytes; but not-yet-identified analytes are undetectable. It is desired, therefore, to have an approach that can detect unknown analytes.
Referring to FIG. 1B, two different types of analytes 140, 150 are dispersed across a substrate 160 by a buffer solvent flow. The analytes 140, 150 are spatially segregated across a surface of the substrate 160, thereby enabling separation of two different analytes 140, 150. The resulting spatial segregation permits detection of individual analytes. Separation in this instance is based on the principle of buffer solvent flow. This approach is low cost, fast, and flexible, but is less specific and less accurate than is desired, and it involves large dimension(s). Another technique might involve molecular migration in a gel (electrophoresis) based on size and molecular weight.
Protein binding to a surface may be affected by the chemical property of the surface. In this way, protein chips with different binding surfaces have been produced. Chromagraphic and spectrographic binding surface technologies have also been evolving, wherein bio-chip detections are typically read by optical methods. When the chip feature (spot) size becomes <1 um, however, optical detection becomes impractical. It is desired to have an approach that enables detection and reading with higher density bio-chips.
Electronic sensors for biomolecule detection have also been demonstrated. Although such electronic sensors have the potential to overcome the spatial limitations of optical detection, electronic sensors by themselves do not appear to obviate the underlying feature size limitations of the polymeric probe-analyte paradigm.
Self aligned monolayers have been demonstrated. The formation of patterned co-planar monolayers (which can be termed ultra thin films) and the use of those patterns to selectively bind colloidal catalysts & plate electroless metals selectively at high resolution are under investigation. Further research into the formation of ultra thin films for the selective adhesion of various types of biological cells is ongoing.
Heretofore, the requirements of a more efficient approach to preparing and immobilizing probes, smaller probe feature sizes, the ability to detect unknown analytes and the detection and reading of higher density bio-chips have not been fully met. It is therefore desired provide techniques that meet these goals.
The embodiments of the invention and the various features and advantageous details thereof are explained more fully with reference to these non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well known starting materials, processing techniques, components and equipment are omitted so as not to unnecessarily obscure the embodiments of the invention in detail. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only and not by way of limitation. Various substitutions, modifications, additions and/or rearrangements within the spirit and/or scope of the underlying inventive concept will become apparent to those skilled in the art from this disclosure.
The descriptions herein of the invention and preferred and alternative embodiments may be better understood in view of the following definitions:
The term “non-polymer” refers to polyatomic organic molecules that do not have repeated units that are either identical or non-identical.
The term “protein chip” refers to a two or three dimensional device that contains immobilized protein species (2 or more proteins) that are arranged in regular patterns or irregular patterns.
The term “optical sensing structure” refers to a device that collects photons from other objects and converts them to electrical signals.
The term “reaction cavity” refers to a 3D space that can hold reactants and allow chemical or biochemical reactions to proceed, typically measured in nanometer or micrometer scales.
The term “feature size” refers to the dimension(s) of an individual feature of a given array. For example, a protein array may have 100 protein spots. Thus the protein spots are the features of the protein array. The dimension of a given spot is the feature size of the spot. It can be measured by area, diameter or lengths of sides.
The phrase “coupling via thiol-based reaction product” refers to covalent bonding formation involving a hydrosulfide group (—SH). It can happen between organic compounds or between a thiol-containing organic compound and metals, such as gold and silver.
A substrate of an apparatus in accordance with a preferred embodiment includes an array of regions defining multiple cells. Each of the cells includes a reaction cavity that contains multiple functional binding groups. The substrate includes a solid material that provides support as well as a functional surface. The substrate can be made up of any of several materials, and preferably inorganic materials such as silicon wafer, glass, metal (aluminum, e.g.), or organic material such as plastic (polycarbonate, e.g.). The surface of the substrate is preferably coated with metal (gold) or a polymer (PEG) or both. Functional groups on the surface may include amine groups or carboxyl groups.
The multiple functional binding groups may be coupled to the substrate via hybridized DNA, a cross-linked polymer, a copolymer, a chain transfer polymer and/or a thiol-based reaction product. The cells preferably each include an analyte sensing structure such as an electrical sensing circuit or an optical sensing structure.
The cells may each comprise a protein chip or gene chip having a feature size preferably between 0.5 microns and 500 microns, and preferably less than approximately 100 microns. The cells may each comprise an electrically-active, combinatorial-chemical (EACC) chip for biochemical analyte detection. The analyte detection may be probe-less. The groups may include non-polymeric components.
The array may include a first density gradient of a first group in a first direction, and may further include a second density gradient of a second group in a second direction. The second direction may be approximately orthogonal to the first direction. Moreover, four significant directions may include, e.g., from an overhead viewpoint, left to right, right to left, up to down and down to up.
The substrate may comprise silicon having a surface modified with silanes, wherein the silanes may comprise phenyl. The multiple groups may include a positively-charged group and a negatively charged group and/or a polar group and a non-polar group.
A method of detecting an analyte uses a substrate including an array of regions defining multiple cells. Each of the cells includes a reaction cavity containing multiple functional binding groups. A channel may be defined between a source and a drain, although not necessarily, or a region may be defined between a pair of electrodes. A voltage is applied between the source and the drain or the pair of electrodes. A parameter indicative of an analyte characteristic is monitored when the voltage is applied. Each of the cells may include an analyte bonded to a self-assembled monolayer to define a channel or region between a source and drain or pair of electrodes, respectively.
A method of making an analyte sensor uses a substrate including an array of regions defining a plurality of cells each including a reaction cavity. Multiple functional binding groups are coupled to each reaction cavity. An analyte sensing structure is formed including the substrate with the array of regions. An analyte is preferably bonded to the multiple functional binding groups of each reaction cavity.
The forming of the analyte sensing structure may include forming a source and a drain for each reaction cavity such that each reaction cavity may define, although not necessarily, a channel between the source and the drain, and coupling a voltage source and monitoring system between the source and the drain, or it may include forming a pair of electrodes for each reaction cavity, and coupling a voltage source and monitoring system between the pair of electrodes. It may also include forming an optical sensing structure.
The method may include modifying a surface of the substrate with silanes, and the silanes may comprise phenyl. Modifications methods may include any of a variety of techniques such as adsorption or charge interaction.
A first gradient of a first group may be formed in a first direction of the array, and a second gradient of a second group may be formed in a second direction of the array. The first and second directions may be orthogonal. Third and fourth directions would include those opposite to the first and second directions.
An apparatus in accordance with an embodiment of the invention includes a substrate that includes an array of regions defining multiple cells, wherein each of the cells includes a reaction cavity that contains multiple functional binding groups. Another embodiment involves a method of detecting an analyte comprising providing a substrate including an array of regions defining multiple cells. Each of the cells includes a reaction cavity containing multiple functional binding groups and defining a channel between a source and a drain or defining a region between a pair of electrodes. In a method in accordance with this embodiment, a voltage is applied between the source and the drain or the pair of electrodes, and a parameter indicative of an analyte characteristic is monitored when the voltage is applied.
Another embodiment includes a process of fabricating an electrically active combinatorial-chemical chip for biochemical analyte detection comprising providing a substrate including an array of regions defining multiple cells each including a reaction cavity. Multiple functional binding groups are coupled to each reaction cavity. In a process in accordance with this embodiment, an analyte is bonded to the multiple functional binding groups of each reaction cavity, and an analyte sensing structure is formed including the substrate with the array of regions. Reaction cavities may be coupled with different functional binding groups or different molecules containing different groups.
To address the problems of creating a large number of specific probes, immobilizing them in small surface areas and applying the chips to samples containing unknown analytes, an embodiment of the invention can adopt a “probe-less” approach. An embodiment of the invention can vary surface properties to selectively attract proteins and/or other molecules. An embodiment of the invention can include creating a binding site with several small molecules (binding components). Small molecules and/or binding components are intended to mean non-polymeric molecules (e.g., can be hetero-oligomers). To achieve this, a limited number of binding components (e.g., groups or molecules, covalently attached or adsorbed) can be used in different ratios and densities to obtain a large number of different chemical matrices that have different binding potentials to different analytes. Biochips made by this method can be termed combinatorial chemical (CC) chips.
An embodiment of the invention can use multiple small compounds (binding components) to assemble arrays of combinatorial chemical matrices for specific analyte binding and detections. Detections can be achieved optically, electronically or electrically. Thus, an embodiment of the invention can eliminate costly and time-consuming specific probe generation and also allow detection of not-yet-identified analytes. An embodiment of the invention is useful for sample profiling, and it is particularly useful for the analyses of proteins as well as other bio-analytes.
Referring to FIG. 1C, a basic element of an AECC chip embodiment of the invention is depicted. A substrate 170 can provide structural support. A first binding group 181 is coupled to the substrate 170. A second binding group 182 is also coupled to the substrate 170 at an inter-molecular distance from the first binding group 171. An analyte 190 binds to both the first binding group 181 and the second binding group 182. The inter-molecular distance between the first binding group 181 and the second binding group 182 corresponds to the inter-molecular distance between the binding locations on the analyte 190. This embodiment of the invention is based on the principle of using different molecules (binding groups) for specific binding of analytes. This embodiment of the invention is very flexible, very compact, sensitive, fast, reasonably specific and accurate. The identification of the analyte depends on the binding pattern of the analyte in the reaction cavities of the apparatus and prior information derived from known analytes
An embodiment of the invention can include: a chip surface divided into multiple sub-areas (regions), each said sub-area can be coated with a combination of different binding components, said binding components can be organic compounds; said different binding components can vary in size, composition, and arrangement of functional groups; the ratios and densities of said binding components can be different among different sub-areas and these sub-areas can be identifiable (indexed) by X-Y coordinators.
In an embodiment of the invention, binding of an analyte on a sub-area can require the presence of 2 or more binding components. An electrical potential can be applied individually to or sensed individually from each sub-area; analyte binding can be detected electrically or electronically; and these detection methods can be used for analyzing (profiling) of biological or chemical samples.
An embodiment of the invention can include a chip having a planar surface with an array of sub-areas; each of the sub-areas can have 1 or more micro or nano-wells (i.e., reaction cavities). Under each such sub-area or well, there can be an electronic sensor and/or electrical structures(s) (e.g., transistors or electrodes for electrical detections). Different chemicals (for instance, 2, 3, 4 or more) can be applied on the surface. When used in different ratios and different densities, a large number of combinations of chemicals (permutations) can be created. A simple way to generate different ratios is to create different gradients from the binding compounds, each of the gradients corresponding to one of the binding components.
Referring to FIG. 2, a multi-chemical-gradients (MCG) chip 200 embodiment of the invention is depicted. The top portion of the figure depicts a top plan view and the bottom portion of the figure depicts a partial cross section view. In this embodiment, A, B C and D are 4 different chemical compounds. As depicted, A-B gradient(s) vary from between right and left and are represented by the horizontal double ended arrow. As depicted, C-D gradient(s) vary from between top and bottom and are represented by the vertical double ended arrow. In this embodiment, a surface 210 of a substrate 220 of the chip 200 includes an array of regions, each of which defines a sub-area 230. Each of the sub-areas 230 includes a sensor unit 240 which in-turn includes a nano-well 250 (reaction cavity) and a semiconductor or electrical sensor 260.
Referring to FIGS. 3A-3C, the chemicals (e.g., compounds of the binding components and solvents/vehicles) can be delivered to the surface of the wells by printing methods. Referring to FIG. 3A, a printing head 310 is coupled to a pair of mixers 320 each of which is in-turn coupled to a pair of reservoirs 330. The printing head 310 can deliver a predetermined ratio of A/B/C/D to a substrate surface 340. Referring to FIG. 3B, a plurality of filled binding cavities 350 is arranged above a plurality of sensors 360 on the substrate. Referring to FIG. 3C, preferably, self-assembled mono-layers (SAM) 370 are formed in each well (reaction cavity 350). For example, the bottom of the well can be coated with gold, a thiol-polyethylene glycol (PEG) derived compound can be used as a base component and compounds with similar (or the same) base structure(s) together with additional functional groups on the other end of the similar base structure molecules are the binding components and used together with base component to form a mixed SAM (self assembled monolayer). The functional groups associated with the binding components play the binding roles in analyte binding.
Referring to FIG. 4, four schematic examples of combinatorial chemical structures are depicted. The organic compounds used as binding components have different functional groups. Positively charged (PC) compounds are typically compounds with amino groups. Negatively charged (NC) compounds can be those containing carboxyl groups, sulfate group and phosphate groups. Compounds which are hydrophobic (nonpolar (NP)) can be those with benzyl ring structures and alkyl chains. Other compounds that are hydrophilic (polar (p)) can also be used, such as compounds with hydroxyl group, amine group, or organic compounds with hetero-atoms (e.g., nitrogen, oxygen). Organic compounds with halogen atoms can also be used. Compounds with reactive compounds groups may also be used, such as compounds with a thiol group, or an aldehyde group. Short peptides, including non-natural amino acids and oligo nucleotides (including those with modified structures) can be used together with other organic compounds. Other factors can also be considered in fabricating CC chips: for example, molecular chain length, position of functional groups, distance between functional groups, number of functional groups per molecule, ratio of mixed functional groups per molecule, arrangement of mixed functional groups on a molecule. These factors are important in generating 3-dimensional binding sites.
Referring to FIG. 5, CC chip can also be made with more than 4 chemical conditions (independent variables). For instance, the binding components (functional groups can be structurally arranged in a molecule to provide a contextual condition. In this way, an additional condition can be molecular chain length. The position of functional groups in a molecule can be another condition. The distance between function groups can be a condition. The number of functional groups per molecule can be a condition. the ratio of mixed functional groups per molecule can be a condition. The arrangement of mixed functional groups in a molecule can also be a condition. Also, the total density of functional groups on surface (region) can be a condition.
Referring to FIGS. 6A-6B, 7 and 8A-8C, different electrical/electronic sensors can be used together with a CC chip. Optical sensors can also be used together with a CC chip. In the case of an active electrical/electronic sensor, the chip can be termed an electrically active CC chip or EACC chip. For example, field-effect-transistor sensors, capacitance and impedance sensors and/or static-electric sensors can be integrated in the chip. Ideally, there is a sensor associated with each reaction cavity and each of the sensors is controlled independently.
Referring to FIG. 6A, a field effect measurement embodiment is depicted. An analyte 610 in a reaction cavity 620 with aqueous buffer is bonded to an SAM layer 630 to define a channel 640 on a substrate 650. The channel 640 is located between a source 645 and a drain 655 which are both coupled to a voltage source and monitoring system 660. Referring to FIG. 6B, a capacitance or impedance measurement embodiment is depicted. The analyte 610 is again bonded to the SAM layer 630 to define the channel 640 on the substrate 650. In this embodiment, the channel 640 is located between a first electrode 670 and a second electrode 675 which are both coupled to a voltage source and monitoring system 660.
Referring to FIG. 7, a co-planar electrode static-electrical or capacitance/impedance measurement embodiment is depicted. A self aligned monolayer 710 is connected to a bottom surface electrode 720. The bottom surface electrode 720 is coupled to a source connector 725. A top surface electrode 730 is located opposite the self aligned monolayer 710 across a reaction cavity with aqueous buffer.
Sample binding: any biochemical or chemical samples can be used, provided chips with affinity surfaces are used. Conditions for sample binding and washing can be similar to those used in standard chromatography procedures: ion exchange, size exclusion, affinity binding, reverse phase binding (e.g., varying pH, ionic strength, solvent concentration) Sample concentrations, binding time and washing conditions can also be modified from the standard procedures. A microfluidic system (or micro electromechanical system (MEMS)) can be combined with the chip.
Detection: Field effect, capacitance and impedance can be monitored for each reaction cavity, provided suitable electrical/electronic structures are made in the chip. An external chip reader is preferably used to collect and analyze the data. FIGS. 8A-8C illustrate an example of detection based on static-electric attraction. After selective binding and washing, an electrical potential is applied between a top surface of the chip 810 and a bottom surface of the chip 820. After drying by vacuum, electrical charges are built up around the molecules. The charges make the molecules move (fly) toward the top surface. Because the top plate can have a transistor (charge detector) corresponding to those in the bottom plate, molecular charging and flying can be regulated and detected independent of those in other reaction cavities.
Several transistors can be in a cell (reaction cavity) with the gates of the transistors coupled to the binding molecules. An SAM layer may not be necessary due to the importance of the distance between the analytes and the gate surface (i.e., the closer the better). The binding of the analytes close to the gate can affect electron distribution and thus the conductance of the transistor (between source and drain). Another type of structure in a cell (cavity) is a combination of electronic sensor (transistor) and electrical sensor (electrode for impedance measurement).
Data interpretation can be based on the premise that no specific probes or binding partners are required. Therefore, data obtained should be compared to reference or control samples or to normalized data. Algorithms can be trained and used to address particular problems.
Embodiments of the invention are applicable to clinical, research, pharmaceutical, agriculture, and environmental protection. Samples may need fractionation or enrichment before contacting a chip. Different chips can be used for the same sample to get complete information of interest.
The invention can include modifying the surface of glass or silicon with silanes that contain phenyl or other aromatic moieties that have absorption at about 260 nm and below. These materials can form a self assembling monolayer (SAM) using standard microelectronics processing techniques such as those used to promote adhesion of photoresists in standard high volume manufacturing (HVM) processing.
Referring to FIG. 9A, an embodiment of the invention is depicted as including a self aligned monolayer (SAM) on a substrate of silicon, silica, or metal oxides. In the case of simple aromatic groups R can be, e.g., hydrogen, amine, ethylenediamine, cyano, methyl, or fluorine groups. Therefore, the starting SAM can have many different chemical characteristics that determine the surface energy, polarity, and capability to attach additional moieties to or just be a relatively inert reaction well characterized starting surface for further modification using deep ultra violet (DUV) light. An embodiment of the invention can use a phenyl group (R═H) for the example depicted in FIGS. 9A-9D.
Once the substrate has been treated it can be exposed (flood or using high resolution mask) on a standard and readily commercially available DUV scanner or stepper. Because the Si—C bond is the weakest bond in the SAM what occurs is the breakage of that bond and the phenyl group is volatilized. In ambient atmosphere, the Si—: radical reacts with O2 & H2O to form SiOH. It is important to note that this is the same surface as the initial substrate surface, but before the formation of the SAM, and it is now one Si atom taller. The dose in mJ/cm2 to completely remove all of the phenyl groups is well documented in several publications and is on the order of 200 to 1000 mJ/cm2 and is also dependent on the type of aromatic and organic group chosen for the original SAM. For instance, it can be assumed that 500 mJ/cm2 is the dose to remove all the phenyl groups.
Referring to FIG. 9B, the resulting surface after the substrate and SAM has been exposed to 50 mJ/cm2 is depicted. After exposure ˜10% of the surface is now available for a 2nd SAM to be formed. It is most important to note in this example that 2nd SAM material may have no aromatic group and, therefore, will not be affected by subsequent DUV exposures because it has substantially no absorption in the DUV spectrum, i.e., above ˜200 nm. In this example, perflourooctyldimethylchlorosilane is used (SAM2) as the next SAM formation material. Treatment of the exposed surface with SAM2 will yield a new surface containing ˜10% of SAM2 and 90% of the original phenyl silane SAM as depicted in FIG. 9C.
In this example, one additional exposure/SAM formation using a trimethoxysilane N-(2-aminoethyl-3-aminopropyl) trimethoxysilane SAM3 is performed, but this process could be continued to build a very large variety of well defined surfaces. In this example a 2nd exposure is 100 mJ and will remove ˜20% of the remaining phenyl groups and following treatment with SAM3 will create a surface with ˜20% SAM3 ˜10% SAM2 and ˜70% of the original SAM as depicted in FIG. 9D.
This procedure could be continued to put more SAMs of known concentration on the surface and subsequent surface chemistry can be done to attach bio-relevant chemistry such as antibodies, DNA, or RNA, to the appropriate R group on the SAM. The surface can thus be patterned in arrays very easily and even have high resolution (<100 nm line/space) within an array. This embodiment of the invention makes it feasible to make arrays of well defined surface chemistry with minimal reticles or masks.
For instance, if it were desired to make small areas (100 um square) of well defined, but different surface concentrations of the three SAMs described above on bare Si metal oxides or glass, the following technique could be used. The surface can be treated with photosensitive trichlorophenylsilane and then exposed via a 10 um×10 um array with dose increments of 5 mJ/cm2 (i.e., ˜1% the does assumed above to be required to remove all the phenyl groups) over a range from 0-500 mJ/cm2. Then SAM2 formation can be performed resulting in 10×10 array containing a ratio of the 1st two SAMs of from approximately 0% to approximately 100% across the array. The 2nd exposure can then be performed but in reverse spatial arrangement of the increments, or with any desired dose range, to yield many different surfaces of known composition. Specifically, with a reverse exposure starting at 500 mJ and going to 0 in the same increments, the result would be 10×10 array that contains ˜100% SAM2 & SAM3, but with ˜0% of the original SAM.
However, in another instance, an embodiment of the invention could utilize the same range and not reverse dose, and this would result in an array with ˜100% original SAM and with the SAM2 & SAM3 increasing in concentration by 1% each until they reach ˜50% each of the surface concentration half way through the array. From then on SAM2 would continue to increase by 1% and SAM3 would decrease by 1% with ˜0% of original same making up the concentration of the surface until 100% SAM2 is reached at last exposure field. The variations on this sub-generic scenario are enormous. The same examples described above can work with 193 nm exposure which will make the aromatic photosensitive SAMs more efficient but will also make many of the non-aromatic SAMs slightly sensitive to each of the subsequent exposures, but since they are so much less absorbing they will be much less involved in the photochemical cleavage, and therefore they are just accounted for in determining the final composition of the surface.
While not being limited to any particular performance indicator or diagnostic identifier, preferred embodiments of the sensor array can be identified one at a time by testing for the presence of sensing with respect to a known concentration of target analyte. The test for the presence of sensing can be carried out without undue experimentation by the use of a simple and conventional impedance spectroscopy experiment. Among the other ways in which to seek embodiments having the attribute of sensing guidance toward the next preferred embodiment can be based on the presence of a characteristic IR spectroscopy signal.
Embodiments of the electrically active combinatorial-chemical chip for biochemical analyte detection can be identified by scanning electron microscope (SEM) cross-sections. Embodiments of the electrically active combinatorial-chemical chip for biochemical analyte detection can also be identified by material analysis of devices containing sensors using techniques such as Auger spectroscopy and/or dynamic secondary ion mass spectroscopy.
Embodiments of the invention can include impedance spectroscopy, amperommetry, voltammetry and other electrochemical techniques used to generate a response from adsorbed analyte through the electrodes/probes. Embodiments of the invention can include the use of optical techniques such as FTIR spectroscopy can be used to identify the functional groups of analyzed chemicals species.
Specific embodiments of the invention will now be further described by the following, nonlimiting examples which will serve to illustrate in some detail various features. The following examples are included to facilitate an understanding of ways in which embodiments of the invention may be practiced. It should be appreciated that the examples which follow represent embodiments discovered to function well in the practice of embodiments of the invention, and thus can be considered to constitute preferred modes for the practice of embodiments of the invention. However, it should be appreciated that many changes can be made in the exemplary embodiments which are disclosed while still obtaining like or similar result without departing from the spirit and scope of embodiments of the invention. Accordingly, the examples should not be construed as limiting the scope of embodiments of the invention.